Work from the Mackall and Gress laboratory and others have shown previously humans regenerate T cells via thymic-dependent pathways or via thymic-independent homeostatic peripheral expansion and that thymic dependent pathways are superior in quality due to their capacity to regenerate a broad T cell receptor repertoire. Unfortunately very few humans who sustain T cell depletion have sufficient residual thymic function to efficiently restore T cells using this pathway. Thus, most patients rely on homeostatic peripheral expansion (HPE) to restore T cell populations and HPE typically results in chronically reduced CD4+ T cell numbers, inverted CD4/CD8 ratios and immune dysfunction. Previous paradigms for understanding T cell homeostasis and pathways of immune reconstitution provided no construct for understanding why humans have such differential capacities to restore CD4+ T cells whereas CD8+ populations are readily restored. A major accomplishment of this project during FY2009 was publication of the results of a series of studies that identified the factors limiting CD4+ immune reconstitution via HPE. Our previous work had demonstrated that lymphopenia in humans results in elevated circulating interleukin-7 (IL-7) levels and current concepts held that elevated IL-7 would drive CD4+ HPE. However, since the relationship between CD4+ counts and IL-7 in lymphopenic humans are consistently inverse, we hypothesized that chronic elevations in IL-7 might serve to diminish CD4+ HPE. To test this, we used lymphopenic mice, and investigated the biology of IL-7 in this setting more closely. We demonstrated that lymphopenic mice also show elevated levels of IL-7 in their tissues that occurs as a result of diminished utilization of IL-7, rather than increased production. We also demonstrated that, despite elevated IL-7 levels, CD4+ T cells do not efficiently expand in this milieu whereas CD8+ T cells do. The breakthrough observation came when we compared CD4+ homeostatic peripheral expansion in lymphopenic mice wherein IL-7 was either exclusively produced by bone marrow derived populations (and not produced by radioresistant host tissues) vs. chimeras wherein IL-7 was produced by both radioresistant tissues and marrow derived populations vs. chimeras wherein IL-7 was exclusively produced by radioresistant tissues but not marrow derived populations. The surprising result was that IL-7 produced by bone marrow derived populations supported efficient CD4+ HPE, whereas production of IL-7 by radioresistant tissues paradoxically diminished CD4+ HPE. Furthermore, elevated IL-7 levels in lymphopenic mice induces downregulation of MHC Class II expression on antigen presenting cell (APCs) populations and diminished IL-7 production by the same APCs. Remarkably, if IL-7 signaling on APCs was inhibited by absence of IL-7 receptor alpha, lack of the common gamma chain receptor or by lack of STAT5, CD4+ HPE was greatly enhanced. Therefore, this work identified an entirely new regulatory axis for controlling CD4+ HPE, which primarily involves IL-7 signaling on APCs. This is paradigm changing since IL-7 signaling on APCs has not previously been shown to be of any real importance, yet this work implicates this axis as a fundamental regulator of CD4+ immune reactivity. This work was published in Nature Immunology in 2009 (Guimond et al, Nat Imm 2009). We are currently extending this work by investigating whether manipulation of IL-7 signaling pathways on APCs could be enhance peripheral CD4+ niches and potentially enhance adoptive immunotherapy of CD4+ T cells or enhance CD4+ mediated antitumor immunity. We also will seek to modulate IL-7 signaling on APCs in the context of autoimmunity to determine whether this axis may be a therapeutic target for new immunosuppressive therapies. Other accomplishments from this project include the demonstration that the thymopoietic effects of keratinocyte growth factor effects require IL-7 and ruled out the possibility that this was due to thymic stromal lymphopoietin, which had remained a possibility given current paradigms (Guimond et al, Blood, 2008 111:969). We also collaborated on a study seeking to investigate role for peripheral NK cells niches in modulating NK cell homeostasis in vivo. Previous work had emphasized that NK cell numbers are modulated by bone marrow production and export, but in a study with Michael Caligiuris group at Ohio State University, we demonstrated that expansion of antigen presenting cells in the periphery of mice leads to dramatic increases in NK cell numbers, which is dependent upon IL15 transpresentation by these APCs (Guimond et al, J Immunol, 2009). Finally, we also published a collaborative report with Michael Bishop identifying a novel relationship between interleukin-7 levels in the blood and the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (SCT). This is important because it could serve as a biomarker for risk for GVHD and also because it lends further credence to the notion that neutralization of IL-7 could protect patients from GVHD after SCT.